The human immunodeficiency virus (HIV) is the etiologic agent of acquired immunodeficiency syndrome (AIDS). Expression of this virus is regulated by several proteins, one of these proteins is TAT which has the ability to autoregulate the expression of HIV by promoting the transcriptional activity of HIV-LTR. The region of HIV-LTR needed for TAT mediated regulation was termed TAR, and the sequence between nucleotide plus 14 to plus 45 relative to the transcription initiation site was shown to be essential for the response to TAT protein. We have previously shown by using in vitro transcription assays that the purified recombinant TAT protein increased the trans-acting activity significantly, but the enhancement of purified TAT protein did not reach the levels obtained with nuclear extract from HeLa cells transformed with TAT gene. The results of gel retardation assay showed an extra band exhibited with HeLa/c TAT nuclear extract. These data suggest the possibility of another important nuclear factor which is only present in HeLa cells transfected by TAT gene. In order to provide more evidence for this cellular nuclear factor's function, we have scaled the HeLa/c TAT cell production from 3 ml to 40 ml pellets of cells and developed a gradient Heparin-Sepharose chromatographic method for fractionation of the HeLa/c TAT nuclear extract. After fractionation we found that different fractions had different gel retardation patterns. The results of in vitro transcription assay showed that only the fractions, which contained the specific binding cellular factor, could significantly increase the trans- acting activity from HIV-LTR promoter. The TAR DNA sequences acted as a strong competitor for the enhancement activity of this factor. In vitro, 40 fold molar excess of TAR competitor relative to the amount of DNA template was able to reduce the trans-activation of the HIV-LTR to the basal level. These results suggest that a specific factor in HeLa/c TAT cells is involved in the TAT protein's function and the effect may be mediated at the DNA level. The presence of a specific cellular factor in HeLa/c TAT cells could provide a new insight into the mechanism of TAT protein's function.